Phytfieldlab kit and the qPCR technics: key for a healthy grass

For decades, the qPCR technique has been a widely used and indispensable technique in both medical and biological laboratories. Today it is popularly known worldwide, the detection technique for COVID-19 and is the key to healthy turf, a novel way to study turf that Tiloom introduces to the turf industry with the innovative Phytfieldlab kit from Microgaia Biotech.

New ways to study the health of our turfgrasses are opening up thanks to qPCR.

New ways of studying the health of our lawns are opening up around the world thanks to the technology put into practice with our new Phytfieldlab Kit.

This technique is based on the exponential amplification of DNA molecules using the enzyme thermostable DNA polymerase. Its acronym is PCR: Polymerase Chain Reaction. It is now an accessible technique so that all fields can be diagnosed quickly and accurately.

Now we can apply this technique to know the phytosanitary status of our sports surfaces, i.e., we can identify pathogenic microorganisms, both in the plant and in the soil, before they produce the disease. This fact allows the greenkeeper to make decisions about the type of treatment to use, assessing the real need for its application, saving costs, optimizing treatments and reducing the presence of these chemical compounds in the soil.

With the Phytfieldlab KIT you can diagnose grass quickly and accurately.

The main advantages are:

• Control and monitoring: allows to anticipate before the first phytopathogenic symptoms appear.
• Prevention: analysis of soil condition prior to the establishment of turf or any crop.
• Certification of pathogen-free turf prior to installation in our stadiums or greens.
• Diagnosis of diseases in both symptomatic and asymptomatic turf: targeted and effective treatments, avoiding the use of broad-spectrum products.
• Wide range detection (substrate, plant, water, seeds).
• Detection and quantification of microorganisms before and after treatment with phytosanitary products, to check their efficacy.
• Quality control of products formulated with beneficial microorganisms.

Differences between a PCR and a qPCR

In the conventional PCR technique, if you want to check whether the DNA has amplified, and therefore whether the microorganism of interest is present in the sample or not, it is necessary to perform another additional technique called electrophoresis. However, even when performing electrophoresis and verifying that the DNA has amplified, at no time is it possible to quantify the DNA that we have amplified, i.e. we cannot know whether we have a high or low amount of the microorganism to be analyzed.

There is a version of the PCR technique, called real-time PCR or qPCR, which allows, in addition to detecting the existence of these microorganisms early, the quantification of them in real time, immediately. Thus, the qPCR technique has a clear advantage over conventional PCR, since it allows us to check the real level of infection by pathogenic microorganisms, and whether their presence in the lawn is relevant or not.

qPCR OPERATION

qPCR is a technique that allows the duplication of DNA from a small amount of this molecule, known as template DNA. This is made possible by an enzyme, DNA polymerase. This technique makes it possible to monitor DNA amplification thanks to the signal produced by a fluorescent marker attached to one of the reaction components, which will provide a light signal of intensity proportional to the amount of DNA being produced. Thus, the more DNA there is, the greater the amount of light emitted by the fluorescent marker.

The emitted signal is intercepted by software that collects the readings on a curve representing the entire amplification reaction cycle.

This equipment is called Thermocycler, and as its name indicates, it is in charge of carrying out the temperature cycles necessary for the DNA amplification reaction to take place. The set temperature cycles are repeated about 40 times, and in each one of them, the following steps take place:

• 1st part of the cycle, temperature 95º. The template DNA molecule is denatured.
• 2nd part of the cycle, temperature 60º. Binding of the primers to the template DNA molecule.
• 3rd part of the cycle, temperature 72°. DNA polymerase elongates the DNA strand from the primers that have been attached to the strand in the previous step by eluting dNTPs.

The highest diagnostic technology available for your field.

In each temperature cycle performed by the thermal cycler, a copy of each of the DNA strands is produced, so that if we start with a new DNA molecule, we will obtain 2 DNA strands in the 1st cycle, 4 DNA strands in the 2nd cycle, 8 DNA strands in the 3rd cycle and so on until the last cycle, where we will have obtained millions of DNA strands.

When DNA amplification occurs in a qPCR reaction, it is possible to see in real time how the DNA molecules are being duplicated. This occurs because one of the components of the reaction, the probe, is labeled with a fluorescent compound, which will emit light only when DNA strand duplication occurs. Thus, the more DNA strands are duplicated, the more light the marker will emit, which will be captured by the thermocycler sensors and finally interpreted by the software.

At the moment when the light generated by the fluorescent compound begins to be significantly visible (indicated by the software by a curve), it means that enough DNA copies of the template DNA have been made for the template DNA to be detected. This moment is known as the detection threshold, which means that from that cycle onwards the detection of template DNA has occurred. In the case that the template DNA comes from a microorganism, it can be said that the detection of that microorganism has occurred.

How do I interpret the information provided by a qPCR?

The information obtained from a qPCR reaction varies depending on the reason for which the reaction was performed.

In the case of detection of plant pathogenic microorganisms, the information being sought is the detection of these pathogens by DNA. It is also extremely useful to know the amount of microorganism that is present in the analyzed sample.

In the event that DNA of the target microorganism is present, during the qPCR reaction, we will be able to see in real time whether DNA duplication occurs thanks to the signal emitted by the fluorescent marker. An exponential amplification line appears at the detection threshold, which, as mentioned above, is the moment at which it can be said that the microorganism is present in the sample.

If the detection occurs in a short time, i.e. in early cycles, it will mean that we have more microorganisms than if the detection occurs in late cycles. This fact is based on the fact that if we start from a greater number of DNA copies, and therefore from a greater number of microorganisms, the necessary DNA copies will be reached earlier so that the thermocycler sensors can detect them.

Tiloom together with Microgaia wants to technify the sports turf sector and with our Phytfieldlab KIT you will be able to go a step further in plant health.